DNA replication is an essential process in the proliferation of both normal and cancer cells. Elucidation of the mechanisms controlling DNA synthesis may yield critical insight into combating the deregulated proliferation exhibited by cancer cells. The initiation of DNA replication during the eukaryotic cell cycle occurs at multiple sites throughout the genome, called origins. The maintenance of genome integrity demands that the DNA be replicated accurately and efficiently, and only once during each cell division cycle. Prior to the DNA synthesis phase of the cell cycle, origins of replication are marked by the formation of a pre-replication complex (preRC). Regulated preRC formation ensures that the DNA is replicated only once per cell cycle. Importantly, the identified protein components of the preRC are conserved among eukaryotes. However, the regulation of preRC components differs between yeast and mammals. Most of the known preRC factors are transcriptionally regulated by the RB/E2F pathway, and are therefore integrated with cell cycle progression. It is the overall goal of this proposal to further characterize the assembly of the mammalian preRC during cell cycle entry. The specific aims of this proposal are: (1) to characterize the functional domains of the requisite preRC component Cdt1, (2) to determine the functional contribution of HBO1 to preRC assembly, and (3) to identify novel preRC factors through co regulated gene expression screening and functional analysis.